High Throughput Monoclonal Antibody Generation by B Cell Panning and Proliferation

ABSTRACT

Provided herein, inter alia, is a method for producing an enriched population of antigen-specific plasma cells. In some embodiments, the method may comprise: (a) obtaining a sample of cells from an animal that has been immunized by an antigen, wherein the sample comprises B cells; (b) enriching for a population of antigen-specific B cells that comprise cell surface antibodies that are specific for the antigen by: i. contacting at least 105 of the cells in said sample, en masse, with the antigen or a portion thereof; and ii. isolating cells that bind to the antigen or portion thereof; and (c) activating the enriched B cells, en masse, in the presence of the antigen or portion thereof, to produce the enriched population of antigen-specific plasma cells.

CROSS-REFERENCING

This application claims the benefit of U.S. provisional application Ser.No. 62/135,084, filed on May 18, 2015, which application is incorporatedby reference herein.

BACKGROUND

The ability of mammals to generate a very diverse repertoire ofantibodies in response to immunization by antigen has been exploited ina wide range of fields, including diagnostics and therapy. Hybridomatechnology was developed by Kohler and Milstein several decades ago andtoday several low-throughput methods for generating monoclonalantibodies have been developed. Such methods include B-cellimmortalization, cloning of antibody-encoding genes and cDNAs bysingle-cell PCR and in vitro “combinatorial” methods that require theproduction huge recombinant antibody libraries.

High throughput antibody-discovery methods have been slow to developbecause, based on sequence information alone, it is impossible todetermine the antigen to which an antibody binds and which heavy andlight chains should be paired together.

Lightwood (J. Immun. Methods 2006 316: 13-143) describes a method forgenerating high-affinity monoclonal antibodies. In Lightwood's method, arelatively small number of B cells from an immunized rabbit are placedinto each well of a multi-well ELISA plate that contains a coating ofsolid phase antigen. After extensive washing to remove non-bound cellsas well as B cells that bound with low affinity, the retained B cellswere cultured for 7 days to induce proliferation and secretion ofimmunoglobulin. Supernatants were screened to identify which wells ofthe plate contain antigen-specific antibodies. Single heavy- and lightchain variable region genes were recovered from individual wells byRT-PCR, and sequenced. The Lightwood method, because it requiresdepositing a relatively small, titrated, amount of B cells into thewells of a 96-well plate, screening supernatants, and performing PCR oncells harvested from individual wells, is inherently low throughput.Moreover, Lightwood's method requires a single activated B cell or arelatively small number of activated B cells per well, because the heavyand light chain pairing would otherwise be unknown. Further, the numberof activated B cells produced using the Lightwood method is insufficientfor the production of hybridomas on a commercial scale.

Reddy (Nat. Biotechnol. 2010 28:965-9) describes a bioinformatics-basedmethod to mine antibody variable region-gene repertoires from bonemarrow plasma cells (BMPCs) of immunized mice. Reddy discovered that theantibody repertoire of bone marrow plasma cells becomes highly polarizedafter immunization, with the most abundant sequences represented atfrequencies between about 1% to over 10% of the total repertoire. Reddypaired the most abundant variable heavy (VH) and variable light (VL)genes based on their relative frequencies, reconstructed them usingautomated gene synthesis, and expressed recombinant antibodies inbacteria or mammalian cells. Antibodies generated in this manner fromsix mice, each immunized with one of three antigens, were mostly antigenspecific (21/27 or 78%). Bone marrow plasma cells are not readilyaccessible, since their isolation requires animal euthanization andselection of CD138⁺ cells. Thus, while some aspects of Reddy's methodcould be considered high throughput, his method is still quite limitedin that it requires a particular type of plasma cell (BMPCs) that areisolated from the bone marrow of a euthanized animal.

Kodituwakku et al (Imm. Cell Biol. 2003 81: 163-170) reviewed the stateof the art in methods for isolating antigen-specific B cells, e.g., bypanning and other similar techniques. Kodituwakku concluded that, whileseveral methods for isolating antigen-specific B cells exist, thosemethods provide very variable results and are plagued by non-specificbinding, which in turn decreases the purity and enrichment of thedesired cells in the isolated population.

In view of the above, there is still a need for high-throughput methodsfor generating antibodies.

SUMMARY

Provided herein, inter alia, is a method for producing an enrichedpopulation of antigen-specific plasma cells. In some embodiments, themethod may comprise: (a) obtaining a sample of cells from an animal thathas been immunized by an antigen, wherein the sample comprises B cells;(b) enriching for a population of antigen-specific B cells that comprisecell surface antibodies that are specific for the antigen by: i.contacting at least 10⁵ of the cells in said sample, en masse, with theantigen or a portion thereof; and ii. isolating cells that bind to theantigen or portion thereof; and (c) activating the enriched B cells, enmasse, in the presence of the antigen or portion thereof to produce theenriched population of antigen-specific plasma cells.

Depending on how the method is implemented, the method can have certainadvantages over conventional methods. For instance, as noted above, mostmethods for isolating antigen-specific B cells result in a population ofcells that contains a significant amount of contaminating cells thatnon-specifically bind to the substrate. The activating step of themethod described herein, only activates B cells that havesurface-tethered antibodies which are bound to the antigen. This methodprovides two effects, firstly, the activating step causes only those Bcells that are specifically bound to the antigen to proliferate, therebyincreasing the relative concentration of those cells relative to thecells that are non-specifically bound to the support. Secondly, theactivating step of the method causes expression of heavy and light chainmRNA to be induced only in those B cells that are specifically bound tothe antigen. In other words, the additional activation step allows oneto selectively stimulate memory B-cell to differentiate and becomeplasmablasts and plasma cells, which are rapidly dividing and expressinglarger amounts of antibody.

On a per cell basis, the enrichment and activation steps, incombination, increase the total number of antigen-specific cells (i.e.,the total number of cells that produce antibodies that bind to theantigen) and, in addition, increases the relative concentration ofantigen-specific cells relative to other cells. The inventors' havefound that up to 25-50% of the collected cells are antigen-specific and,as such, the enrichment may be well over 100-fold in many cases. Assuch, the collected cells can be screened (e.g., using conventionalhybridoma methods) much more efficiently than other populations ofcells, e.g., splenocytes or the like. Further, the enrichment andactivation steps, in combination, may activate B cells that might nototherwise be activated in vivo (the reasons for which are unknown)thereby increasing the diversity of the pool of antibodies available foranalysis. Finally, the enrichment and activation steps, in combination,cause “rare” antigen-specific B cells (e.g., B cells that encodeantibodies that are unrelated by lineage to other antibodies beingproduced by the population) to proliferate, thereby increasing theprobability that those cells (or sequences produced by the same) areidentified when the cell population (or a collection of sequencesobtained from the same) is screened.

Moreover, because the cells are employed en masse in the present method,the method is highly scalable and can be tailored, without additionaleffort, to produce as many different antigen-specific plasma cells asdesired. For example, in some cases, a representative portion of thefull antigenic response of an animal can be surveyed and thousands, ifnot tens of thousands, of hybridomas or sequences that encodeantigen-specific antibodies can be obtained without additional effort.The ability to screen a significant portion of the full antigenicresponse of an animal also allows one to identify “rare” antibodies(e.g., antibodies that are not very abundantly expressed in the contextof an otherwise strong immune response) and to perform further analysison the antigenic response, such as lineage analysis or an analysis ofthe abundance of different antibody sequences. Lineage analysis requiressequence information for as many antibodies as possible and is usefulbecause, once a candidate antibody that has desirable activity (e.g., apotential therapeutic or diagnostic activity) has been identified, thenanalysis of the lineage of that antibody can provide an insight into thestructure of the antibody and allows one to identify amino acid residuesthat can be substituted (see, e.g., Yu et al, PLoS ONE, 2010 5: e9072).This, in turn, provides a way that second generation antibodies can becreated in a targeted manner. Further, the magnitude of an immuneresponse varies greatly from animal to animal and epitope to epitopeand, in some cases, the titer of antibody to a particular epitope may beextremely low. The present method, because it applies an en masseapproach, accommodates the unpredictability of an animal's immuneresponse and facilitates the identification of rare antibodies becausethe entire immune response can effectively be screened in a singleexperiment.

These advantages are, in practice, impossible to achieve using theLightwood method described above because Lightwood's methods requirealiquoting a relatively small number of antibody-producing cells intoeach well of a microtiter plate in the hope that some of the wellsreceive no more than a single antigen-specific B-cell that becomesactivated. Lightwood's method is limited by: a) Poisson distribution,meaning that there will always be wells that contain no antigen-specificB cells in addition to wells that contain too many antigen-specific Bcells, making the method inefficient; b) lack of scalability in that themethod requires an impractical amount of work (e.g., potentiallyhundreds or thousands of microtiter plates, assays and PCR reactions) tosurvey the entire immune system of an animal; and c) the lack of anefficient way to accommodate the unpredictability of an animal's immuneresponse. The lack of an efficient way to accommodate theunpredictability of an animal's immune response means that, even underthe best of conditions, practice of Lightwood's method requires doingthe same experiment several times using several different dilutions ofcells in order to obtain a dilution that potentially works, and thenperforming the method.

In addition to the above, the enrichment and activation steps alsoactivates antibody expression in each of the activated B cells. This, inturn, vastly increases the amount of mRNA encoding antigen-specificantibodies in the resultant cell population. The additional activationstep is particularly relevant for high throughput embodiments thatinvolve sequencing the heavy and light chain cDNAs of the collectedcells because, after enrichment, the enriched cells are still highlyimpure. The “noise” is generated by B cells that are non-specificallycaptured on the support or not washed away. These cells, which aremostly plasma cells, express several-fold more antibody thanantigen-specific memory B cells. Therefore, without activation, the VHand VL sequences from the contaminating cells are often more abundantthan the sequences for the antigen-specific antibodies. Therefore,sequencing the VH and VL cDNAs from a population that has been enrichedbut not activated (after bead purification for example), generates adata set that is predominantly composed of sequences for antibodies thatare not antigen-specific. In contrast, sequencing the VH and VL cDNAsfrom a population that has been enriched and activated, generates a dataset that is predominantly composed of sequences for antibodies that areantigen-specific.

As will be described in much greater detail below, the population ofcells made by the present method may be fused with a suitable fusionpartner to make hybridomas (which may be screened by hybridoma) or insome embodiments, cDNAs encoding the VH and VLs may be sequenced fromthose cells.

In hybridoma embodiments, the collected cells may fused with a suitablefusion partner to make hybridomas, and the hybridomas may be screenedusing any convenient method (e.g., ELISA) to identify a hybridoma thatproduces an antigen-specific antibody. In these embodiments, performingthe enrichment and activation steps vastly decreases the number ofclones to be screened (by over 10-fold in some instances), and, at thesame time increasing the number of cells being input into the fusionstep. Additionally, because the “rare” B cells have been amplified inthe previous steps, there is a higher chance those cells will berepresented in the hybridoma population. Specifically, the inventorsfound that “rare” antibodies, i.e., antibodies that are at low abundancewould not otherwise be sampled from a B cell population (i.e., B cellsobtained directly from an animal, without enrichment or activation)without an exhaustive sequencing effort or an exhaustive screen ofhybridomas made from those cells, appear to be at an increasedconcentration relative to other cells in the enriched and activated Bcells. As such, such rare antibodies have a higher chance of beingidentified by sequencing the heavy and light chains of an enriched andexpanded B cell population, or by screening hybridomas made from thosecells. Finally, immortalizing the collected B cells allows one toproduce as much antibody as necessary without having to clone, sequenceand validate in transient assay. This can save a significant amount oftime and minimize overall handling in a production context.

In sequencing embodiments, the most abundant sequences (e.g., those thathave a read count above a threshold, e.g., a threshold in the range of5-10 sequence reads) can be readily identified. These sequences are morelikely to be antigen-specific and, as such, the present method allowsthe rapid identification of hundreds, if not thousands, ofantigen-specific VH and VL sequences without implementing alabor-intensive screening effort. As will be described in greater detailbelow, the sequences can be analyzed by tanglegram analysis to identifypairs of VH and VL sequences that can be expressed in a cell and tested.In addition, because multiple sequence reads are obtained for each VH orVL, sequencing errors can be corrected using bioinformatic methods.

Moreover, the inventors unexpectedly found that if enrichment andactivation are performed the heavy and light chain sequences obtained bysequencing the population of activated cells are sometimes polarized andin certain cases can be paired by their relative abundance. In theinventor's experience, the heavy and light chain sequences obtained fromB cells (i.e., B cells obtained directly from an animal, withoutenrichment or activation) are not as polarized and cannot be paired bytheir abundance. This allows one to identify antibodies by: enrichingfor a population of memory B cells by affinity to an antigen, activatingthose cells, collecting the cells (without any screening of culturesupernatant to identify which cells produce antigen-specificantibodies), sequencing the heavy and light chains separately, andidentifying and pairing heavy and light chain sequences together bytheir relative abundance to produce antibodies that bind to the antigen.This method, which can be done using a sample that in many cases can bevery easy to obtain removes a significant bottleneck from theconventional methods.

In addition, the enrichment step of the method provides a means by whichantigens can be multiplexed and “application-specific” antibodies can beproduced. In multiplexing embodiments, an animal may be immunized withmultiple antigens, and antigen-specific cells for each of a plurality ofantigens may be enriched separately from one another. Theantigen-specific cells can then be activated and collected separatelyfrom one another, as summarized above, to provide a plurality ofpopulations of cells, each specific for a different antigen.“Application-specific” antibodies, i.e., antibodies that are selectedfor a particular use, e.g., FACS, immunoprecipitation,immunohistochemistry, therapeutics, etc.) can be produced by tailoringthe enrichment step to favor cells that produce a particular type ofantibody (e.g., antibodies that bind to one antigen but not another,antibodies that recognize cells, antibodies that bind only under certainconditions, or antibodies that bind to an antigen in fixed tissuesection, etc.).

These and other advantages may be may be apparent in light of thedescription that follows below.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 schematically illustrates some of the general principles of thepresent method.

FIG. 2 schematically illustrates an embodiment in which the activated Bcells are fused with a fusion partner to make hybridomas.

FIG. 3 schematically illustrates how lineage trees can be aligned in atanglegram.

FIG. 4 schematically illustrates how intervening VH and VL sequences inan anchored tanglegram can be paired.

FIG. 5 schematically illustrates a way for resolving an ambiguouslyranked light chain sequence.

FIG. 6 schematically illustrates how application-specific B cells can beenriched.

FIGS. 7A and 7B show a comparative analysis of VH sequences recoveredfrom the NGS of total PBMC versus B-cells after antigen specific panningand proliferation (BPP). FIG. 7A: overlap between total PBMC andaffinity selected/proliferated VH sequences. FIG. 7B: Unique VHsequences only found in affinity selected/proliferated dataset.

FIG. 8 shows an example of an anchored tanglegram.

FIG. 9 shows a characterization of a cell culture by flow cytometry.

FIG. 10 shows polarization in abundance numbers between AS (enriched andactivated) and PBMC samples.

DEFINITIONS

Before the present subject invention is described further, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantibody” includes a plurality of such antibodies and reference to “aframework region” includes reference to one or more framework regionsand equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The term “plurality” refers to more than 1, for example more than 2,more than about 5, more than about 10, more than about 20, more thanabout 50, more than about 100, more than about 200, more than about 500,more than about 1000, more than about 2000, more than about 5000, morethan about 10,000, more than about 20,000, more than about 50,000, morethan about 100,000, usually no more than about 200,000. A “population”contains a plurality of items.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. These terms are well understood by those in the field, and referto a protein consisting of one or more polypeptides that specificallybinds an antigen. One form of antibody constitutes the basic structuralunit of an antibody. This form is a tetramer and consists of twoidentical pairs of antibody chains, each pair having one light and oneheavy chain. In each pair, the light and heavy chain variable regionsare together responsible for binding to an antigen, and the constantregions are responsible for the antibody effector functions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Theantibodies may be detectably labeled, e.g., with a radioisotope, anenzyme which generates a detectable product, a fluorescent protein, andthe like. The antibodies may be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies mayalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termare Fab′, Fv, F(ab′)₂, and or other antibody fragments that retainspecific binding to antigen, and monoclonal antibodies.

Antibodies may exist in a variety of other forms including, for example,Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e. bi-specific) hybridantibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987))and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), which are incorporated herein by reference). (See, generally,Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed., 1984, andHunkapiller and Hood, Nature, 323, 15-16, 1986).

An immunoglobulin light or heavy chain variable region consists of aframework region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs have been precisely defined (see,“Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S.Department of Health and Human Services, 1991). The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs. The CDRs are primarilyresponsible for binding to an epitope of an antigen.

By “variable region of an immunoglobulin chain” or an “immunoglobulinchain variable region” is a polypeptide comprising at least a portion ofthe variable domain of a heavy (i.e., the VH domain) or a light chain(i.e., the VL domain) of an immunoglobulin, where the portion of the VLand the VH domains form an antigen binding domain of an immunoglobulin.Thus, the variable region of an immunoglobulin includes three CDRsflanked by either or both of FR1 and FR4 (e.g., FR1, CDR1, FR2, CDR2,FR3, CDR3, FR4). In some embodiments, the immunoglobulin chain variableregion is the region on one of either the heavy or the light chainwhich, when combined with the immunoglobulin chain variable region ofthe other chain (i.e., the light or the heavy chain) of immunoglobulin,forms the antigen binding domain.

By “antigen binding domain” is meant the region of a single heavy chainassembled with a single light chain in an immunoglobulin, which has thespecific binding activity of the intact antibody for its specificantigen. Thus, an intact IgG immunoglobulin, which comprises two heavychains and two light chains, has two antigen binding domains.

The term “natural” antibody refers to an antibody in which the heavy andlight chains of the antibody have been made and paired by the immunesystem of a multi-cellular organism. Spleen, lymph nodes, bone marrowand blood are examples of tissues that contain cells that producenatural antibodies. For example, the antibodies produced by B cellsisolated from a first animal immunized with an antigen are naturalantibodies. Natural antibodies contain naturally-paired heavy and lightchains.

The term “naturally paired” refers to heavy and light chain sequencesthat have been paired by the immune system of a multi-cellular organism.

The term “mixture”, as used herein, refers to a combination of elements,e.g., cells, that are interspersed and not in any particular order. Amixture is homogeneous and not spatially separated into its differentconstituents. Examples of mixtures of elements include a number ofdifferent cells that are present in the same aqueous solution in aspatially undressed manner.

The term “assessing” includes any form of measurement, and includesdetermining if an element is present or not. The terms “determining”,“measuring”, “evaluating”, “assessing” and “assaying” are usedinterchangeably and may include quantitative and/or qualitativedeterminations. Assessing may be relative or absolute. “Assessing thepresence of” includes determining the amount of something present,and/or determining whether it is present or absent.

The term “enriched” is intended to refer to component of a composition(e.g., a particular type of cells) that is more concentrated (e.g., atleast 2×, at least 5×, at least 10×, at least 50×, at least 100×, atleast 500×, at least 1,000×), relative to other components in the sample(e.g., other cells) than prior to enrichment. In some cases, somethingthat is enriched may represent a significant percent (e.g., greater than2%, greater than 5%, greater than 10%, greater than 20%, greater than50%, or more, usually up to about 90%-100%) of the sample in which itresides.

The term “enriching” is intended to any way by which antigen-specificcells can be obtained from a larger population of B cells. As will bedescribed in greater detail below, enriching may be done by panning,using bead or cell sorting, for example.

The term “obtaining” in the context of obtaining an element, e.g., cellsor sequences, is intended to include receiving the element as well asphysically producing the element.

The term “peripheral blood mononucleated cells” or “PBMCs” refers toblood cells that have a single approximately round nucleus (as opposedto a lobed nucleus) and includes lymphocytes (T cells, B cells and NKcells), monocytes and macrophage. PBMCs can be enriched from whole bloodusing a ficoll gradient.

The term “cell surface antibody” refers to an antibody that is tetheredto the surface of a B cell. B cells that have cell surface antibodiesinclude memory B cells and naïve B cells. Such an antibody may bereferred to as the “B cell receptor” in some publications.

The term “specific binding” refers to the ability of an antibody topreferentially bind to a particular antigen that is present in ahomogeneous mixture of different molecules. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable molecules in a sample, in some embodiments more than about10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

In certain embodiments, the affinity between an antibody and an antigenwhen they are specifically bound in a capture agent/analyte complex ischaracterized by a K_(D)(dissociation constant) of less than 10⁻⁶ M,less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻⁹ M,less than 10⁻¹¹ M, or less than about 10⁻¹² M or less.

The term “antigen-specific B cells” refers to memory B cells that havean antibody that specifically binds to an antigen on their surface, aswell as progenitors thereof.

A cell is “derived from” a host if the cell, or the progeny thereof, wasobtained from the host. The progeny of a progenitor cell are derivedfrom the progenitor cell.

The term “support comprising the antigen” comprises any type of support(e.g., a solid or semi-solid support, including plates and beads) thatcontains an antigen, or a portion thereof, immobilized thereon. Anantigen may be immobilized on a support directly or indirectly, e.g.,via a linker, via a biotin-streptavidin interactin or via a cell, forexample. Methods that enrich for antigen-specific B cells by panning orusing beads make use of such a support.

The term “panning” is used to refer to a method by which B cells areapplied to a container (e.g., a plate) that has one or more surfacesthat are coated in an antigen or portion thereof. Unbound cells can beremoved by washing the surface after the cells are applied to it.

The term “bead-based enrichment” is used to refer to a method by which Bcells are mixed with beads, e.g., magnetic beads, that are linked to anantigen or portion thereof.

The term “cell sorting” is used to refer to a method by which B cellsare mixed a detectable antigen (e.g., a fluorescently detectableantigen) in solution. In cell sorting methods, cells that are bound tothe antigen are sorted from the unbound cells. Fluorescence-activatedcell sorting (FACS) is an example of a cell sorting method.

The term “antigen, or a portion thereof” refers to an antigen that wasused for immunization, or part of the same (e.g., a peptide of 5-20amino acids in length).

The term “complex immunogen” is intended to refer to an immunogen thatcontains a plurality of antigens. A complex immunogen can be composed ofa plurality of different antigens that have been separately made andthen mixed together, or they may be naturally complex (e.g., as is thecase when one uses an entire cell or a fraction thereof) in animmunization.

The term “activating” is referred to the stimulation of B cells to a)proliferate and b) differentiate into plasmablasts and/or plasma cellsand c) secrete antibodies. B cell activation can be done by contactingthe B cells with antigen, T cells expressing CD40L and cytokines,although other methods are known (see, e.g., Wykes, Imm. Cell. Biol.2003 81: 328-331).

The term “activated B cells” refers to a cell population that comprisesthe progeny of a B cell that was activated. As noted above, activationcauses B cells to proliferate, and the progeny of such cells arereferred to herein as activated B cells.

The term “collecting” refers to the act of separating the cells that inthe culture medium from the a substrate. Collecting may be done bypipetting or by decanting, for example.

The term “immunized by an antigen” and grammatical equivalents there of(e.g., “immunized animal”) is intended to refer to any animal (humans,rabbits, mice, rats, sheep, cows, chickens, humans, camels) that ismounting an immune response an antigen. An animal may be exposed to aforeign antigen via exposure to an infectious agent, a vaccination, orby administrating an antigen and adjuvant (e.g., by injection), forexample. The term “immunized by an antigen” is also intended to includeanimals that are mounting an immune response against a “self” antigen,i.e., have an autoimmune disease.

The term “en masse” refers to the addition of a sample to a container asa single unit, without sub-fractionating or sub-dividing the samplebeforehand or afterwards. For example, aliquoting portions of a sampleinto individual wells of a multi-well plate is not an en masse action.Aliquoting portions of a sample into the same well of a multi-well plate(i.e., making multiple transfers from one container to another using thesame pipettor) is an en masse action. At least 10⁵, at least 10⁶ or atleast 10⁷ cells may be used en masse.

The terms “ranking” and “ranked order of abundance” refer to the orderof sequences when they are listed by their abundance, i.e., with themost abundant sequence first, the second most abundant sequence next,and the third most abundant sequence next, and so on. In certain cases,sequences may be ranked by making a frequency distribution, and thenordering the sequences by their frequency.

The term “corresponding rank” or “correspondingly ranked” refer to twosequences that have the same positions in two ranks. For example, thefirst, second and third positions in a first rank correspond to thefirst, second and third positions in a second rank, respectively. Aswill be described in greater detail below, ambiguities in a ranking(e.g., if two sequences are expressed at a similar abundance) can may beresolved by analyzing the lineage of those sequences.

As used herein, the term “lineage-related antibodies” and “antibodiesthat related by lineage” as well as grammatically-equivalent variantsthere of, are antibodies that are produced by cells that share a commonB cell ancestor. Antibodies that are related by lineage bind to the sameepitope of an antigen and are typically very similar in sequence,particularly in their L3 and H3 CDRs. Both the H3 and L3 CDRs oflineage-related antibodies can have an identical length and a nearidentical sequence (i.e., differ by up to 5, i.e., 0, 1, 2, 3, 4 or 5residues). In certain cases, the B cell ancestor contains a genomehaving a rearranged light chain VJC region and a rearranged heavy chainVDJC region, and produces an antibody that has not yet undergoneaffinity maturation. “Naïve” or “virgin” B cells present in spleentissue, are exemplary B cell common ancestors.

Related antibodies are related via a common antibody ancestor, e.g., theantibody produced in the naïve B cell ancestor. The term “lineagerelated antibodies” is not intended to describe a group of antibodiesthat are not produced by cells that arise from the same ancestor B-cell.A “lineage group” contains a group of antibodies that are related to oneanother by lineage.

As used herein, the term “at least the CDR3s” or “at least the CDR3sequences” refers to only CDR3 sequences, CDR3 sequences in conjunctionwith CDR1 and/or CDR2 sequences or a sequences of at least 50 contiguousamino acids of the variable domain, up to the entire length of thevariable domain, where the sequence contains a CDR3 sequence.

As used herein, the terms “cladogram” and “lineage tree” refers to adiagram, resulting from a cladistic analysis, which depicts ahypothetical branching sequence of lineages leading to the individualspecies of interest. The points of branching within a cladogram arecalled nodes.

As used herein, the term “constructing a phylogenetic tree” refers tothe computational act of making a phylogentic tree from sequences.

As used herein, the term “lineage” refers to a theoretical line ofdescent.

As used herein, the term “lineage analysis” refers to the analysis ofthe theoretical line of descent of an antibody, which is usually done byanalyzing a lineage tree.

As used herein, the term “sequence read” refers to a sequence ofnucleotides determined by a sequencer, which determination is made, forexample, by means of base-calling software associated with thetechnique.

As used herein, the term “clade” refers to a group of VH or VL sequencesthat are related by lineage, i.e., they are descents of a commonancestor.

As used herein, the term “antibody heavy and/or light chain sequences”refers to either the VH of an antibody, the VL chain of an antibody, orboth the VH and VL chains of an antibody.

As used herein, the term “obtaining the amino acid sequences” refers toobtaining a file containing amino acid sequences. As is well known, anucleic acid sequence can be translated into an amino acid sequence insilico.

As used herein, the term “most abundantly expressed”, with reference toa protein sequence, refers to a protein sequence that is most abundantin a sample. The abundance of a protein can be determined by, e.g.,counting sequence reads encoding that protein. The protein with the mostsequence reads is the most abundant protein.

As used herein, the term “sequence comparison” refers to a method forcomparing a query sequence with a database of sequences, and identifylibrary sequences that resemble the query sequence above a certainthreshold. BLAST (Altschul et al Journal of Molecular Biology 1990 215:403-10) and FASTA (Lipman et al Science 1985 227: 1435-41) are examplesof algorithms that can be used for sequence comparison, and many othersare available.

As used herein, the term “tanglegram” refers to a pair of lineage treesin which leaves are aligned with one another in a way that minimizes“crossovers” in the pairings (see FIG. 3). Every branch of a lineagetree is rotatable around a node and, in making a tanglegram, thebranches are been rotated around their nodes to provide the best matchbetween the leaves, where the best match minimizes the number ofcrossovers between the matched leaves. Tanglegrams have been widely usedin biology to compare evolutionary histories of things that are relatedto one another, e.g., to analyze the molecular evolution of host andparasite species, or to analyze genes of species in the samegeographical area. Tanglegrams are described in great detail inScornavacca et al (Bioinformatics 2011 27: 248-256), Venkatachalam et al(IEEE/ACM Trans Comput Biol Bioinform. 2010 7: 588-97) and Lozano(IEEE/ACM Trans Comput Biol Bioinform. 2008 5:503-13). FIG. 3 shows anexample of how tanglegram analysis can align two simple lineage trees.In this example, the crossovers are completely eliminated.

Tanglegrams can be “anchored” using leaves that are known to pair withone another. In these embodiments, the branches are rotated around theirnodes until there is a minimal number of cross-overs (e.g., nocrossovers) between the anchored sequences. After the trees have been“aligned” by tanglegram analysis, the leaves that are known to pair canbe connected by an edge (as indicated by lines drawn between the leavesof the phylogenetic trees illustrated in FIGS. 4 and 8, for example). Ifthe leaves that are known to pair are connected by an edge, theintervening leaves, in theory, can pair with one another as long as theydo not create a cross-over event with an edge or one another. FIGS. 4and 8 show examples of an anchored tanglegram in which the interveningsequences (i.e., the sequences that are not linked by an edge) can bepaired with one another as long as the pairing does not create across-over with an edge or one another.

As used herein, the term “aligned”, in the context of a tanglegram,refers to two sequences that lie across from one another in atanglegram, where “across from one another” means that they can bepaired without making a cross-over with a known edge. One sequence inone lineage tree may be aligned with more than sequence in another tree.The tanglegram shown in FIG. 8 shows an example of a tanglegram of a VHand VL sequences are aligned.

As used herein, the term “inputting” is used to refer to any way ofentering information into a computer. For example, in certain cases,inputting can involve selecting a sequence or a model that is alreadypresent on a computer system. In other cases, inputting can involveadding a sequence or a model to a computer system. Inputting can be doneusing a user interface.

As used herein, the term “executing” is used to refer to an action thata user takes to initiate a program.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

One implementation of the present method is schematically illustrated inFIG. 1. With reference to FIG. 1, certain embodiments of the method maycomprise: obtaining a population of cells that comprise B cells from ananimal that has been immunized by an antigen. In the example shown inFIG. 1, peripheral blood mononucleated cells (PBMCs) are obtained.However, other sources of B cells (e.g., spleen, lymph nodes and bonemarrow) may be used instead. Next, the method comprises enriching for apopulation of antigen-specific B cells that comprise cell surfaceantibodies that are specific for the antigen. In general terms, thisstep of the method involves contacting at least 10⁵ of the cells in thesample, en masse, with the antigen or a portion thereof and isolatingcells that bind to the antigen or portion thereof. This step may be doneby panning (as shown in FIG. 1), although any other suitable method,e.g., bead-based enrichment method, or by cell sorting (e.g., FACS), maybe used instead. For example, in embodiments that use panning or beads,this step of the method may be done by i. contacting at least 10⁵ of thecells in the sample, en masse, with a support (e.g., a container orpopulation of beads) comprising the antigen, or a portion thereof, underconditions by which the antigen-specific B cells bind to the antigen orportion thereof; and ii. washing the support to remove unbound cells. Inembodiments that use cell sorting, this step of the method may be doneby: i. contacting at least 10⁵ of the cells in the sample, en masse,with the antigen, or a portion thereof under conditions by which theantigen-specific B cells bind to the antigen or portion thereof; and ii.sorting cells that bind to the labeled antigen or portion thereof.

Next, the enriched B cells are activated, en masse, in the presence ofthe antigen or portion thereof. In these embodiments, the antigen orportion thereof may be added to culture medium in addition to the othernecessary ingredients for activating the cells. Finally, the method maycomprise collecting the activated cells, e.g., by decanting or pipettingthe cells.

As will be described in greater detail below, the collected cells may beused in a variety of different ways without the need for testing theculture medium of the activated cells to determine if the cells secretean antibody that binds to the antigen or portion thereof.

In some embodiments, the enriching step of the method comprises i.contacting a plurality of cells from the sample (e.g., at least 10⁵, atleast 10⁶ or at least 10⁷ cells), en masse, with a support comprisingthe antigen, or a portion thereof, under conditions by which theantigen-specific B cells bind to the antigen or portion thereof; and ii.washing the support to remove unbound cells. The enrichment step maydone using any suitable enrichment method that employs a support, e.g.,an antigen-coated solid support (such as a Petri dish or the like) orantigen-coated beads (e.g. magnetic beads or streptavidin coated beads).These methods may sometimes be referred to as “panning” or “bead-based”enrichment and examples of such methods are described are described inLightwood supra, Kodituwakku, supra, and U.S. Pat. No. 7,790,414 whichare incorporated by reference. In alternative embodiments,antigen-specific B cells may be obtained by FACS (see, e.g., Weitkamp etal 2003 J. of Imm. Methods 275, 223-237 and US20130017555). If FACS isused, the sorted cells may be all combined into a single wells, notseparated into different wells. In sorting embodiments, the cells may belabeled to detect the presence of other cell surface markers, therebyallowing specific cell types, e.g., memory cells, to be enriched.

In performing this step of the method, the B cells may sometimes beallowed to contact a support (e.g. a plate or beads if beads are beingused) that has been coated in antigen or portion thereof for sufficienttime to allow binding. B cells that do not bind to the support may thenbe removed, leaving those B cells which are bound to the support. Insome embodiment, the antigen is the same as that used to immunize theanimal, or a portion thereof. As will be described in greater below, theantigen on the substrate may be presented to the B cells in a way thatselects B cells that express application-specific antibodies (i.e.,antibodies that can be used for FACS, immunohistochemistry, westernblotting, therapeutics, etc.). In certain embodiments, the B cells maybe depleted for cells that non-specifically bind to other antigens. Inthese embodiments, the cells may be bound to a first substrate to removecells that non-specifically bind to another antigen, prior to binding tothe support containing the antigen of interest. This step can be used toremove cells that bind to antigens that are similar to the antigen ofinterest, or other sources of potential contamination. In other cases, asecond antigen can be added to the solution during the enrichment step,where the second antigen blocks non-specific binding of some cells tothe antigen on the substrate.

Once the B cells have been in contact with the substrate for sufficienttime to allow binding, the mixture is then washed with a medium thatfacilitates removal of the non-adhering cells from the substrate butwhich leaves cells that are bound to the support via antibodies that areon the surface of the B cells. Suitable media will be known to thoseskilled in the art or can be readily determined empirically by thoseskilled in the art. Any culture medium for example Roswell Park MemorialInstitute medium (RPMI) or Dulbecco's Modified Eagle Medium (DMEM) maybe used. In some cases, a number of washes may be employed to remove thenon-adherent cells, e.g., 5 or 10 or more washes.

The enriching may be done in any suitable container. The container usedmay be chosen to accommodate the volume of cells (e.g., PBMCs) used inthe method. In some cases, 10⁴ to 10⁷ cells (e.g., 10⁵ to 10⁷ or morecells) in a volume of 0.5 ml to 50 mls (e.g., 1 ml to 10 ml) aredeposited into a vessel that contains the support. In some embodiments,the antigen or portion thereof will saturate the surface of thecontainer. Those skilled in the art will be readily able to adjust theparameters of the enrichment step to optimize the number and type of Bcells that are retained on the substrate. Parameters which may beadjusted include the volume added to the substrate, the surface area ofthe substrate, the concentration or amount of antigen bound to thesubstrate; the concentration or amount of B cells added to thecontainer; the source of B cells (e.g. if the B cells are from a lowresponder then more B cells could be used); the number of washes toremove the non-adhering cells; the wash solution, etc. In contrast tosome other methods, this method does not require depositing single cellsinto multiple containers, nor carefully titrating the number of B cellsadded to a container in the expectation that each container will containa relatively small number, e.g., under 5 (e.g., one, two, three, four orfive) antigen-specific B cells. Rather, in the present method as many Bcells as desired can be added to the substrate, without titrating theamount of antigen-specific B cells are being added to the container.

Alternatively, the antigen or portion thereof may be coated onto beads.The use of beads to select for cells which bind to an antigen ofinterest is well documented in the art. Briefly, for example, theantigen or portion thereof may be bound to magnetic beads. The B cellsare then mixed with the magnetic beads and those B cells which bind tothe antigen or portion thereof will bind to the magnetic beads via thecapturing agent. The B cells which bind to the magnetic beads may thenbe obtained by magnetic separation. The use of magnetic beads isdescribed in Lagerkvist et al. (BioTechniques 1995 18:862-869).

As noted above, cell sorting (e.g., FACS) may be used in someimplementations of the method. In these implementations, the antigen orportion thereof may be fluorescently labelled to facilitate the FACSsorting of the B cells. If cell sorting is used, the majority, e.g., atleast 50%, at least 70% or at least 90% of the enriched cells, e.g., atleast 1,000, at least 5,000, at least 10,000, at least 50,000 or atleast 100,000 cells may be activated, en masse, in the next step of themethod.

When enriching for those cells that produce an antibody thatspecifically binds to the antigen of interest, it may be desirable toensure that B cells that non-specifically bind (e.g. to the support orto cells not expressing the antigen) are not selected. In theseembodiments, the B cells may be first exposed to the container and/orsupport to which no antigen or portion thereof has been bound and thendisposing of those B cells which non-specifically bind to the container.Similarly, if beads are used, then prior to incubating the B cells withantigen-coated beads the B cells may first be incubated with uncoatedbeads and the cells that bind to the uncoated beads may then be removed.Alternatively, cells that non-specifically bind to the antigen orportion thereof can be removed after to the cells after they have beenenriched.

As would be apparent, the cells used in the present method may beobtained from various sources. For example, the cells could be obtainedfrom the spleen, lymph nodes bone marrow or peripheral blood of ananimal that has either been immunized with an antigen, or that hasdeveloped an immune response to an antigen as a result of disease.Animals may be immunized with a selected antigen using any of thetechniques well known in the art suitable for generating an immuneresponse (see Handbook of Experimental Immunology, D. M. Weir (ed.), Vol4, Blackwell Scientific Publishers, Oxford, England, 1986). Manywarm-blooded animals, such as humans, rabbits, mice, rats, sheep, cows,chickens, humans, camels or pigs may be immunized. In some embodiments,the animal may have an autoimmune disease, or may have developedresistance to or has recovered from a disease (e.g., cancer). In someembodiments, antibody-producing cells may also be obtained from asubject that has generated the cells during the course of a selecteddisease or condition. For instance, antibody-producing cells from ahuman with a disease of unknown cause, such as rheumatoid arthritis, maybe obtained and used in an effort to identify antibodies which have aneffect on the disease process or which may lead to identification of anetiological agent or body component that is involved in the cause of thedisease. Similarly, antibody-producing cells may be obtained fromsubjects with disease due to known etiological agents such as malaria orAIDS. These antibody-producing cells may be derived from the blood,lymph nodes or bone marrow, as well as from other diseased or normaltissues. Cells obtained from humans that have been exposed to anantigen, e.g., vaccinated, may be used.

In some embodiments, the animal may be been immunized with the antigen,e.g., multiple times in the presence of an adjuvant. In theseembodiments, suitable antigens are numerous and include soluble andsolubilized proteins, including extracellularly-exposed fragmentsmembrane proteins In particular embodiments, the animal may be immunizedwith a complex immunogen that contains multiple antigens (e.g., at least2, at least 5, at least 10, at least 50, at least 100, at least 500 orat least 1,000, up to 5,000 or more antigens).

If PBMCs are used, they may be enriched from blood that has been treatedwith an anticoagulant such as heparin or EDTA. PBMCs may be isolatedfrom whole blood by lympholyte density centrifugation (Biozol; #CL5120)or using a Ficoll density gradient (Sigma-Aldrich, catalog number:10771; MP Biomedicals, catalog number: 091692254). Methods for isolatingPBMCs are well known (see, e.g., Panda, Bio-protocol 2013 3: e323) andin certain cases may include the following steps: collect venous bloodsample and mix with heparin, layer the blood on the top of FicollHistopaque, centrifuge 30 min at 100×g in 4° C. in a swing-out bucket;remove the cells in the interphase between histopaque and medium, andthen wash the cells, e.g., with PBS. In many species, the approximateyield of cells from 4 ml of blood may vary between 10⁵-10⁸.

After the antigen-specific B cells have been enriched, they areactivated in the presence of the antigen or portion thereof. The B cellsmay be activated by any suitable method, e.g., by CD40 activation. Incertain cases, the activating may be done by, e.g., by contacting theimmobilized cells with a medium containing the antigen or portionthereof that also contains CD40-L (which may be on a T cell) and one ormore cytokines and/or growth factors (see, e.g., Liebig et al, J VisExp. 2010 Mar. 5 37: 1734; van Kooten et al, J. Leukoc. Biol. 200067:2-17; Kondo et al, Clin Exp Immunol. 2009 155:249-56; WO 91/09115; WO94/24164; Tsuchiyama L et al., Hum Antibodies. 1997 8:43-7; Imadome etal., Proc Natl Acad Sci. 2003 100:7836-40, among others).

In some embodiments, the activation step results in activation of atleast 100 antigen specific B cells (e.g., at least 500 antigen specificB cells, at least 1,000 antigen specific B cells, at least 5,000 antigenspecific B cells, at least 10,000 antigen specific B cells, at least50,000 antigen specific B cells or at least 100,000 antigen specific Bcells) in the same vessel, resulting in a culture medium that contains amixture of at least 10⁵ activated B cells (e.g., at least 10⁶ activatedB cells, at least 10⁷ activated B cells or at least 10⁸ activated Bcells) that is made up of several clonal populations of B cells (e.g.,at least 50 clonal populations of B cells, at least 100 clonalpopulations of B cells, at least 500 clonal populations of B cells, atleast 1000 clonal populations of B cells, at least 5000 clonalpopulations of B cells, at least 10,000 clonal populations of B cells,at least 50,000 clonal populations of B cells or at least 100,000 clonalpopulations of B cells, etc.), and a highly complex mixture ofantibodies. The next step of the method may be performed without testingthe culture medium to determine if antigen-specific antibodies are beingproduced although, in some cases, one may want to optionally test theculture medium for antigen-specific antibodies before continuing.

After the B cells are activated and have proliferated, they arecollected. As noted above, this may be done by pipetting or decantingthe culture medium from the solid support, and placing the decantedmaterial into a vessel. This step may be done en masse, too. Thecollected B cells may be used in a variety of applications as describedbelow.

B Cell Fusion Embodiments

In some embodiments, the method may comprise fusing the activated cellswith a fusion partner to produce a plurality of hybridomas and screeningthe hybridomas to identify a hybridoma that produces an antibody thatbinds to the antigen or portion thereof.

In these embodiments, the collected cells may be fused with a suitableimmortal cell (e.g., NIH 3T3, DT-40 or 240E cell, etc.; Spieker-Polet etal, Proc. Natl. Acad. Sci. 92: 9348-9352, 1995) to produce hybridomas.In these embodiments, at least 10⁵, at least 10⁶ or at least 10⁷ of thecollected B cells may be fused with a suitable fusion partner anddeposited into wells, using any suitable method. Supernatants from thewells are screened for antibody secretion by enzyme-linked immunosorbentassay (ELISA) and positive clones secreting monoclonal antibodiesspecific for the antigen can be selected and expanded according tostandard procedures (Harlow et al. Antibodies: A Laboratory Manual,First Edition (1988) Cold spring Harbor, N.Y.; and Spieker-Polet et al.,supra). Suitable monoclonal antibodies may be further selected in thebasis of binding activity, including its binding specificity, bindingaffinity, binding avidity, a blocking activity or any other activitythat causes an effect (e.g. promoting or inhibiting a cellularphenotype, e.g., cell growth, cell proliferation, cell migration, cellviability (e.g., apoptotis), cell differentiation, cell adherence, cellshape changes (e.g., tubular cell formation), complement dependantcytotoxicity CDC, antibody-dependent cell-mediated cytotoxicity ADCC,receptor activation, gene expression changes, changes inpost-translational modification (e.g., phosphorylation), changes inprotein targeting (e.g., NFκB localization etc.), etc., or inhibition ofreceptor multimerization (e.g., dimer or trimerization) orreceptor-ligand interactions). Antibody-encoding nucleic acids areisolated from hybridomas using standard molecular biology techniquessuch as polymerase chain reaction (PCR) or reverse transcription PCR(RT-PCR) (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed.,Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.), andtransferred to a different host to express recombinant antibodies. Anexample of this method is illustrated in FIG. 2.

Sequencing Embodiments

In some embodiments, the method may comprise making cDNA from thecollected cells, and sequencing the cDNA to obtain a plurality of VH(heavy chain variable domain) sequences and a plurality of VL (lightchain variable domain) sequences. In these embodiments, the method mayfurther comprise selecting a VH and a VL sequence, and testing anantibody comprising the selected sequences to determine if the antibodybinds to the antigen or portion thereof. In some embodiments and as willdescribed in greater detail below, the heavy and light chain sequencesmay be selected by: i. obtaining a tanglegram of a plurality of the mostabundant heavy and light chain sequences, wherein the tanglegram isanchored using heavy and light chains that are naturally paired with oneanother; ii. selecting a heavy chain sequence and a light chainsequence, wherein the selected heavy and light chain sequences arealigned with one another in the tanglegram; and iii. testing an antibodycomprising the selected heavy and light chain sequences to determine ifthe antibody binds to the antigen or portion thereof.

In these embodiments, cDNAs encoding the antibodies produced by thecollected cells may be sequenced. In certain embodiments, at least thecDNAs encoding the variable domains of the heavy and light chains areamplified. Strategies for performing RT-PCR to amplify sequences thatencode antibodies for rabbits, mouse and humans, among others, aredescribed in US20040067496, Kantor et al (Ann. N Y Acad. Sci. 1995 764:224-7), Boekel et al (Immunity. 1997 7:357-68), Yamagami et al (Immunity1999 11:309-16), Beerli et al (MAbs. 2010 2), Morbach et al (Mol.Immunol. 2008 45:3840-6), Kiippers et al (Methods Mol Biol. 2004 271:225-238) and Seidl et al (Int. Immunol. 1997 9:689-702), which areincorporated by reference herein. Several strategies for cloningantibody sequences by PCR are known and may be readily adapted for usein the instant method (e.g., by using a CDR-specific primer in additionto a disclosed primer). Such strategies include those described by:LeBoeuf (Gene. 1989 82:371-7), Dattamajumdar (Immunogenetics. 199643:141-51), Kettleborough Eur. J. Immunol. 1993 23:206-11), Babcook(Proc. Natl. Acad. Sci. 1996 93: 7843-7848) and Williams (Cold SpringHarb. Symp. Quant. Biol. 1989 54:637-47) as well as many others. Incertain cases, the second primer may be a mixture of different primersor degenerate primers, for example.

In some embodiments, the entire polynucleotide encoding a VH or VLsequence may be amplified using primers spanning the first and lastcodons of those regions. In certain cases, universal primers ordegenerate primers may be used. Suitable tails may be added to theprimers to facilitate sequencing. Amplification procedures using nestedprimers may also be used, where such nested primers are well known toone of skill in the art.

As would be apparent, the sequencing may be done using a next generationsequencing platform, e.g., Illumina's reversible terminator method,Roche's pyrosequencing method (454), Life Technologies' sequencing byligation (the SOLiD platform), Life Technologies' Ion Torrent platform,of Pacific Biosciences SMRT platform, etc. Examples of such methods aredescribed in the following references: Margulies et al (Nature 2005 437:376-80); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9);Shendure (Science 2005 309: 1728); Imelfort et al (Brief Bioinform. 200910:609-18); Fox et al (Methods Mol Biol. 2009; 553:79-108); Appleby etal (Methods Mol Biol. 2009; 513:19-39) and Morozova (Genomics. 200892:255-64), which are incorporated by reference for the generaldescriptions of the methods and the particular steps of the methods,including all starting products, reagents, and final products for eachof the steps. In other embodiments, the sequencing may be done usingnanopore sequencing (e.g. as described in Soni et al Clin Chem 53:1996-2001 2007, or as described by Oxford Nanopore Technologies).

Depending on the read depth desired, the sequencing step may result inat least 5,000 heavy chain sequence reads (at least 10,000, at least50,000, at least 100,000, at least 500,000, at least IM, at least 5M, atleast 10M, at least 50M or at least 100M heavy chain sequence reads) andat least 5,000 light chain sequence reads (at least 10,000, at least50,000, at least 100,000, at least 500,000, at least IM, at least 5M, atleast 10M, at least 50M or at least 100M heavy chain sequence reads). Incertain embodiments, each of the sequence read can cover the entirevariable region of the heavy chain or light chain of an antibody. Atthis point, the heavy and light chain sequences are unpaired in thesense that one does not know which light chain sequence pairs with whichheavy chain sequence.

As noted above, the heavy and light chains may be paired usingtanglegram analysis. In these embodiments, the method may comprisemaking a lineage tree for the most abundant VH sequences and a lineagetree for the most abundant VL sequences and aligning the trees in atanglegram. In some embodiments, the number of sequences that areselected for analysis may be chosen arbitrarily or, in certainembodiments, the number of sequences that are chosen for analysis may berepresented by a certain number of sequence reads (e.g., at least 10, atleast 50, at least 100, at least 500, at least 1,000, at least 2,000, atleast 5,000 or at least 10,000 sequence reads). Either way, a number ofthe most abundant VH and VL sequences (e.g., up to 50, up to 100, up to500, up to 1,000, up to 2,000, or up to 5,000 of the most abundant VHand VL sequences) are selected. After the most abundant VH and VLsequences are selected, a tanglegram is constructed, which generalinvolves making separate lineage trees (one tree for the selected VHsequences and another tree for the selected VL sequences) and thenmaking a tanglegram that is anchored by VH and VL that are known to pairwith each another. As explained above, in these embodiments, thebranches of the lineage trees are rotated around their nodes until thereare a minimal number of cross-overs (e.g., no crossovers) between theanchored sequences. After the trees have been “aligned” by tanglegramanalysis, the sequences that are known to be paired to each other can beconnected by an edge, as shown in FIGS. 4 and 8. The interveningsequences can be paired with one another and tested as long as thepairing does not create a cross-over with an existing edge. This conceptis illustrated in FIG. 4. The lineage analysis may be done using atleast the CDR3 regions (e.g., only the CDR3 regions, all of the CDRs, orthe entire variable region) encoded by the sequence reads, for example.In some cases, sequence abundance may be taken into consideration inselecting a VH sequence and a VL sequence for testing. For example, ifthere are three intervening VH sequences and three intervening VLsequences, then the most abundant sequences are more likely to pair withone another.

As noted above, the tanglegrams can be anchored using VH and VL that areknown to pair with each another. These sequences may be obtained usingany suitable method. For example, VH and VL sequences may be amplifiedfrom individual B cells (obtained by plating the collected B cells at asingle-cell dilution) by single-cell RT-PCR. Alternatively, thecollected B cells can be fused with a suitable fusion partner (asdescribed above) and the VH and VL sequences can be amplified from ahybridoma. The number of sequences used for anchoring a tanglegram mayvary depending on the number of sequences being analyzed. In someinstances, on average, at least 10%, at least 20%, or at least 30% or atleast 50% of the sequences in a tanglegram should be anchored. Dependingon the size of the tanglegram, this may be represented by at least 10,at least 20, at least 30, at least 50 or least 100 or more edges. Incertain embodiments, one may need to obtain at least 100, at least 500,at least 1,000, at least 5,000, at least 10,000 or more VH and VLsequences that have known pairing.

In the sequence data obtained using the present method, there is often adefined number of antibodies, i.e., approximately 10-500 VH and VLsequences, that are much abundant than the rest and that can be readilyranked by their abundance (see FIG. 10). In some embodiments, this“polarization” often allows one to pair the VH and VL sequences by theirabundance and produce antibodies that bind to the original antigen. Assuch, in certain embodiments, after the sequences have been obtained,the VH and VL sequences can be independently ranked in accordance withtheir abundance (i.e., with the with the most abundant sequence first,the second most abundant sequence next, and the third most abundantsequence next, and so on). This ranking step may be based on thesequence reads alone where, for example, all sequence reads that areidentical or very similar (i.e., at least 98% or 99% identical toaccommodate sequence errors) are placed into a group and the number ofsequence reads for each group is counted. The ranking stem may also bedone using translated sequences, where the sequence reads are grouped byat least the CDR3 regions (e.g., only the CDR3 regions, all of the CDRs,or the entire variable region) encoded by the sequence reads. In thisexample, all sequence reads that encode antibodies with similar oridentical CDR3 regions, CDRs or variable regions (i.e., at least 98% or99% identical or having one or two amino acid substitutions toaccommodate sequence errors) are placed into a group and the number ofsequence reads for each group is counted.

After the ranking is done, the heavy and light chains may be pairedtogether in accordance with their ranked order of abundance. In thisexample, the most abundantly ranked heavy chain sequences are pairedwith a correspondingly ranked light chain sequence, i.e., the first,second, third, fourth and fifth most abundant heavy chain sequence arepaired with the first, second, third, fourth and fifth most abundantheavy chain sequence. Antibodies containing such paired heavy and lightchains may be made and tested. Rank ordering is described inUS20110312505, Haessler (Methods Mol. Biol. 2014 1131:191-203) and Reddy(Nat Biotechnol. 2010 28:965-9).

In certain cases, the rank order of two or more sequences may beambiguous because they have a similar abundance (e.g., an abundance thatis within 10% or 20% of one another). For example, the most abundantheavy chain sequence may be represented by 10,000 sequence reads,whereas the second and third most abundant heavy chain sequences may berepresented by 5,500 and 6,000 reads respectively. In this example, thesecond and third heavy most abundant chain sequences may have anambiguous ranking and, as such, can be difficult to determine if theyare correctly ranked.

Ambiguous rankings can be resolved by analyzing the lineages of thesequences that are ambiguously-ranked and the lineage of thecorrespondingly ranked sequences from the other chain. This “local”lineage analysis method generally involves constructing two separatelineage trees showing the theoretical relationship of the most abundantheavy and light chain sequences (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 ormore of the most abundant sequences) with other, similar, sequencesrepresented in the sequencing data. One lineage tree is constructedusing the VH sequences and the other lineage tree is constructed usingthe VL sequences. In attempting to resolve an ambiguity in the rankingof two light chains, one can look at the phylogenetic tree of the heavychain (in which the correspondingly ranked sequences are not ambiguous),and determine which order the two light chains should be ranked. Forexample, if two heavy chain sequences map to a clade that contains arelatively large number of sequence that differ from one another by oneor two amino acids (which have resulted from affinity maturation), thenthe corresponding light chain sequences are likely to map to a cladethat contains a relatively large number of sequence that differ from oneanother by one or two amino acids. In another example, if one heavychain sequence maps to a clade that contains a relatively large numberof sequence that differ from one another by one or two amino acids andthe other heavy chain sequences maps to a side branch off that clade,then the corresponding light chain sequences are likely to map to asimilar clade and a side branch off that clade. In another example, iftwo heavy chain sequences map to different branches in a highly branchedclade, then the corresponding light chain sequences likely map todifferent branches in a highly branched clade. In another example, if aheavy chain sequence maps to a basal clade (a clade that is near theroot of the tree), then the corresponding light chain sequence likelymaps to a basal clade. Therefore, in attempting to resolve potentialambiguities in a ranking of, e.g., a VH sequence, one can look at thephylogenetic tree of the VL sequences (in which the correspondinglyranked sequences are not ambiguous), and determine which ranking iscorrect, based on where the correspondingly ranked sequences are on thattree.

This concept is illustrated in FIG. 5. In FIG. 5, the second and thirdmost abundant light chain sequences are ranked ambiguously. By comparingthe lineage tree for the most abundant light chain sequences to thelineage tree for the most abundant heavy chain sequences, one canresolve the ranking of the light chain sequences and determine which onecorrectly pairs with which heavy chain. In this example, the second andthird most abundant light sequences pair with the second and third mostabundant light chain sequences in some combination. Based on the VH andVL trees, one can deduce that overall, the pairing is likely to becorrect because the branching pattern of the heavy and light chain treesis similar; b) based on the heavy chain tree, the second most abundantlight chain sequence likely is likely to be in the same clade as themost abundant heavy light sequence, and c) based on the heavy chaintree, the third most abundant light chain sequence is likely to be in aside branch of clade that contains the second most abundant light chainsequence. Based on this analysis, the ambiguity of the ranking can beresolved. In this case, the most abundant heavy chain pairs with themost abundant light chain, the second most abundant heavy chain pairswith the second most abundant light chain, and the third most abundantheavy chain pairs with the third abundant light chain. Even if suchanalysis does not remove all ambiguities, the candidate pairings can benarrowed down and tested.

In certain embodiments, the method may involve (a) obtaining a sample ofcells from an animal that has been immunized by an antigen, wherein thesample comprises B cells; (b) enriching for a population ofantigen-specific B cells that comprise cell surface antibodies that arespecific for the antigen by: i. contacting at least 10⁵ of the cells inthe sample, en masse, with the antigen or a portion thereof; and ii.isolating cells that bind to the antigen or portion thereof; and (c)activating the enriched B cells, en masse, in the presence of theantigen or portion thereof, to produce the enriched population ofantigen-specific plasma cells; (d) making cDNA from the enrichedpopulation of antigen-specific plasma cells; and (e) obtaining aplurality of heavy chain variable domain sequences and a plurality oflight chain variable domain sequences by sequencing the cDNA; and (f)comparing the sequences obtained in (e) to a plurality of heavy chainvariable domain sequences and a plurality of light chain variable domainsequences obtained by sequencing a second portion of the sample of (a).This implementation of the method may be used to identify sequences thattruly encode antigen-specific antibodies, to identify antibodies thatare produced by B cells that have potentially not been activated in theoriginal sample, without the need to perform a screen.

As noted above, the method may be multiplexed in that an animal may beimmunized with complex immunogen, and antigen-specific cells for each ofa plurality of antigens of the immunogen may be enriched separately fromone another. In these methods, two, three, four, five or more, at least10, at least 20, at least 50, or at least 100 or more different supportsmay be used to separately isolate the different antigen-specific Bcells, thereby allowing a single animal to be immunized with a compleximmunogen. B cells that are specific for each antigen of interest can beenriched separately from one another. In some embodiments, theantigen-specific cells can then be activated and collected, assummarized above, to provide a plurality of populations of cells, eachspecific for a different antigen. These methods generally involve: (a)obtaining a sample of cells from an animal that has been immunized by ancomplex immunogen, wherein the cells comprise B cells; (b) enriching fora first population of antigen-specific B cells that comprise cellsurface antibodies that are specific for a first antigen by: i. bindingat least 10⁵ of the cells in said sample, en masse, with the firstantigen or a portion thereof; and ii. isolating cells that bind to thefirst antigen or portion; (c) activating the enriched B cells, en masse,in the presence of the first antigen or portion thereof to obtain afirst population of activated B cells; and (d) collecting the firstpopulation of activated B cells from the support. This method maycomprise (b) enriching for a second population of antigen-specific Bcells that comprise cell surface antibodies that are specific for asecond antigen of the immunogen by: i. binding at least 10⁵ of the cellsin said sample, en masse, with the second antigen or a portion thereof;and ii. isolating cells that bind to the second antigen or portionthereof; (c) activating the enriched B cells, en masse, in the presenceof the second antigen or portion thereof to obtain a second populationof activated B cells; and (d) collecting the second population ofactivated B cells activated cells from the support.

Also as noted above, the enrichment step may be tailored to enrich forapplication-specific antibodies, i.e., antibodies that are selected fora particular use, e.g., FACS, immunohistochemistry, therapeutics, etc.).Examples of this are shown in FIG. 6. In some embodiments, theantigen-specific B cells may be enriched by binding to the antigen undera particular condition, e.g., a particular salt concentration, pH, undera particular mechanical force, temperature, or in the presence of anadditive to the binding buffer. In other embodiments, theantigen-specific B cells may be captured using antigens that arepresented in a particular orientation (which should make the finalantibody more suitable for ELISA assays and the like), fixed/embeddedantigen (which should make the final antibody more suitable for IHCassays done on FFPE samples), beads that are linked to the antigen(which should make the final antibody more suitable forimmunoprecipitation) and cells (which should make the final antibodymore suitable for FACS and cell enrichment applications). These are onlyexamples and others would be apparent.

Once the heavy and light chain sequences are paired, polynucleotidesencoding the variable regions of the antibodies are made and expressed.In some cases, the synthesized sequences may be inserted intoappropriate vectors for expression, for example, as full length IgGs orsingle chain antibodies, by transfection of HEK293 cells or anothersuitable cell type. The nucleic acids, once made, can be operably linkedto an expression polynucleotide that will allow for expression, andoptionally secretion of a functional antibody from a host cell. Inparticular cases, the expressed antibody may be a single chain antibody.Strategies for producing a recombinant antibodies, e.g., in mammalian,bacterial and yeast host cells are well known. Once an antibody moleculeof the invention has been produced, it may be purified by any methodknown in the art for purification of an immunoglobulin molecule, forexample, by chromatography (e.g., ion exchange, affinity, particularlyby affinity for the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. In manyembodiments, antibodies are secreted from the cell into culture mediumand harvested from the culture medium.

After a recombinant antibody is produced by another host cells, it maybe tested in a variety of assays and, depending on how the antibody isgoing to be used, it may be humanized. For example, an antibody may betested in a binding assay (e.g., an ELISA, a FACS assay or usingimmunohistochemistry) or an activity assay (which may be in vivo, invitro or in a cell-free system), methods for which are well known (see,e.g., US20040067496).

An antibody produced by the instant methods finds use in, for example,diagnostics, in antibody imaging, and in treating diseases treatable bymonoclonal antibody-based therapy. In particular, an antibody humanizedby the instant methods may be used for passive immunization or theremoval of unwanted cells or antigens, such as by complement mediatedlysis or antibody mediated cytotoxicity (ADCC), all without substantialimmune reactions (e.g., anaphylactic shock) associated with many priorantibodies.

In one embodiment, a humanized version of an identified antibody isprovided. In certain cases, humanized antibodies may be made bysubstituting amino acids in the framework regions of a parent non-humanantibody to produce a modified antibody that is less immunogenic in ahuman than the parent non-human antibody. Antibodies can be humanizedusing a variety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). In certain embodiments, framework substitutions areidentified by modeling of the interactions of the CDR and frameworkresidues to identify framework residues important for antigen bindingand sequence comparison to identify unusual framework residues atparticular positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann etal., Nature 332:323 (1988)). Additional methods for humanizingantibodies contemplated for use in the present invention are describedin U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417;5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT publications WO98/45331 and WO 98/45332. In particular embodiments, a subject antibodymay be humanized according to the methods set forth in published U.S.patent applications 20040086979 and 20050033031. Accordingly, theantibodies described above may be humanized using methods that are knownin the art.

In one embodiment of particular interest, a subject antibody may behumanized in accordance with the methods set forth in great detail inU.S. Pat. No. 7,462,697 which application is incorporated by referencein its entirety. In general, this humanization method involvesidentifying a substitutable position of an antibody by comparingsequences of antibodies that bind to the same antigen, and replacing theamino acid at that position with a different amino acid that is presentat the same position of a similar human antibody. In these methods, theamino acid sequence of a parental antibody is compared to (i.e., alignedwith) the amino acid sequences of other antibodies that are clonallyrelated to the parental antibody to identify variation tolerantpositions. The amino acid sequence of the variable domain of theparental antibody may be compared to a database of human antibodysequences, and a human antibody that has an amino acid sequence that issimilar to that of the parental antibody is selected. The amino acidsequences of the parental antibody and the human antibody are compared(e.g., aligned), and amino acids at one or more of the variationtolerant positions of the parental antibody are substituted bycorrespondingly positioned amino acids in the human antibody. In thishumanization method, the CDR regions of the antibody may be humanized inaddition to the framework regions.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain embodiments and aspects of the present invention andare not to be construed as limiting the scope thereof.

Overview

One of the challenges in the application of next generation sequencing(NGS) technology to monoclonal antibody development is to identify,among millions of VH/VL sequences, the subset representing targetedantigenic response. Integration of experimental design withbioinformatics (data mining strategies) is essential to achieve thisobjective. Described herein is a new approach that combines B cellenrichment, activation and NGS to identify antigen-specific IgG withhigher confidence and better resolution.

B-cells derived from the peripheral blood of an immunized animal can beused as the starting material. This sample contains a large populationof B-cells of different differentiation stage and specificity, immature,naïve and plasma B-cells and it is estimated (based on published flowcytometry analysis) that only 0.1-1% of the circulating B-cells arerelated to the antigenic response following immunization. To unravelthis complexity, narrow the scope to antigen-specific B-cells andincrease sensitivity (sequencing depth), B-cells are selected on solidsupport for affinity to the antigen. In-vitro proliferation promotescell division and differentiation of antigen-specific B-cells, which arecollected and sequenced. The transition of an antibody-presenting B-cell(which should in theory be memory B cells) into an antibody-producingB-cell is accompanied by a strong increase in IgG mRNA expression, whichcan be detected by NGS and their abundance can be estimated by countingsequence reads. In addition, only the B-cells initially activated byinteraction with the antigen differentiate into plasma cells.

Depending on how the method is implemented, the method provides greatersensitivity, a reduction in “noise” and is highly scalable, unlike othermethods. Greater sensitivity is achieved by enriching forantigen-specific B-cells: enrichment on antigen coated plates may allowfor a 5-10× enrichment in B cells expressing antigen-specific IgG, andselecting for B cells that express rare monoclonal antibodies that wouldotherwise be missed without enrichment. A reduction is noise can beachieved by activating the antigen-specific B-cells. In this step, onlythe B-cells that are bound to the antigen proliferate and differentiateinto plasma cells. The significant increase in mRNA expression from theactivated population of cells helps distinguish those sequences fromnoise that is inherent in more traditional selection methods (e.g.,sequences from plasma cells that non-specifically bind duringenrichment). Finally, the method is highly scalable in that a largepopulation of cells (e.g., of 5-10 million cells) can be subjected toenrichment and used in the method.

Example 1 Protocol for PBMC Isolation, Enrichment and Activation 1)Antigen Coating and Blocking:

-   -   1. Sterile filter the antigen solution by using a 0.22 μm        syringe filter.    -   2. Coat a high affinity dish with antigen (1.5 ug/ml) in 10 mL        of PBS.    -   3. Incubate overnight at 4° C.    -   4. Aspirate the plate the next morning and wash 2× with 10 ml        PBS.    -   5. Block the plate by adding 10 ml 5% FBS/PBS solution. Incubate        for 2 hours at room temperature or 30 minutes at 37° C.    -   6. Aspirate the plate and then wash 1× with 10 ml 1640 RPMI        media

2) PBMC Isolation:

-   -   1. Add 15 ml of gradient media (Ficoll-Paque Premium 1.084) into        a Leucosep lymphocyte separation tube. It should be above the        white membrane. Heavily shake the Ficoll bottle before use.    -   2. Centrifuge at 1200 rpm for 30 seconds. The gradient should be        below the white membrane.    -   3. Dilute the rabbit blood 1:1 with room temperature PBS. Then        pour 20 mL of the diluted rabbit blood into the Leucosep tube.        It should layer on top of the membrane and gradient media.    -   4. Centrifuge at 1500 rpm for 30 min.    -   5. Using a Pasteur pipette, carefully transfer the PBMC layer        into a new 50 ml tube (be careful to avoid mixing the PBMC layer        with the serum layer).    -   6. Wash with 45 ml of DPBS and centrifuge at 1150 rpm for 10 min        to collect the cells. Do not aspirate the supernatant as you may        aspirate cells, instead pour it off into a waste container.    -   7. Repeat step 6.    -   8. Resuspend the cell pellet in 5 ml B-cell fusion media and        count the number of viable cells by hemocytometer or FACS.

3) Panning:

-   -   1. Add 5M PBMCs to a high affinity dish (coated, blocked, and        washed) with 5 ml B-cell fusion media (cell density 1 M/ml). If        cell number lower than 5M, put all the cells into 5 ml.    -   2. Incubate at 37° C. for 90 min with slow agitation (50 rpm).    -   3. Aspirate the media and wash 2× with of 5 ml B-cell fusion        media. Add the media slow, and always to the same spot on the        dish. Check the dish with a microscope after each wash. If the        number of attached cells is low, reduce the number of wash        steps; if cell number is high and there are many cells are in        suspension then wash one more time. Two washes are standard.

4) B-Cell Culture:

-   -   1. After washing, add 10 ml of B-cell fusion media in the dish.    -   2. Then add 10 ml CD40L feeder cell media in the dish.    -   3. Incubate at 37° C. and 5% CO₂.    -   4. After 5-6 days use a microscope to check the cell status and        number, if media is yellow and many cells can be seen under the        microscope, then add 10 ml of B-cell fusion media to the dish        and do the fusion on the 7^(th) day; if cells grow up and expand        and media is still pink, do the fusion on the 7^(th) day; if        cell number is low, add 10 ml Feeder cell media (without feeder        cell) and do the fusion on the 10^(th) day.

Example 2 Protocol for Hybridoma Production 5) B-Cell Fusion

-   -   1. Harvest the B-cells into a 50 ml tube.    -   2. Count the number of live cells with trypan blue or FACS.    -   3. Transfer 8 M B-cells to another new 50 ml tube and add 4 M        240E-W2 into the tube (if the cell number is less than 8M, then        add 240E cell number at a ratio of 2:1).    -   4. Centrifuge at 1500 rpm for 5 min.    -   5. Aspirate the supernatant completely while being careful to        not lose any cells.    -   6. Add 0.3-0.4 ml of pre-heated PEG, slowly (should take        10-30 s) to the bottom of the tube using a sterile Pasteur        pipette (should take 1 min total).    -   7. Slowly add 21 mL of pre-heated 1640 RPMI media (should take 1        min).    -   8. Slowly add 21 mL of pre-heated B-cell growth media (should        take 1 min).    -   9. Closed tube cap, invert the tube slowly to mix the medias.    -   10. Add feeder cells (240E-W2) into the tube (2×10⁶ of 240E per        1×96 plate).    -   11. Spin down the cells at 1100 rpm for 5 min.    -   12. Aspirate the supernatant.    -   13. Add 1 ml of 240E growth media and resuspend the cell pellet        with a transfer pipette.    -   14. Dilute the cells with B-cell growth media and add into 4        96-well plates (100 ul/well).    -   15. After 24 hours, add 2× HAT 240E-W2 media to the plates (100        ul/well).

Example 3 Preparation of VH and VL Libraries

B-cells from peripheral blood were enriched and activated according tothe protocol described above. 5 million cells were collected 5-8 daysafter proliferation, the cells were then lysed in TRIzol reagent (LifeTechnology) and total RNA was isolated using RNeasy purification kitaccording to the manufacturer's protocol (Qiagen). RNA concentration wasmeasured with an ND-1000 spectrophotometer (Nanodrop). A total of 2 ugof total RNA was combined with TCL buffer at a 1:8 v/v ratio, aliquot in2 wells (one for each VH and VL library), and mRNA was then isolated byhybridization to well-immobilized oligo-dT according to manufacturer'sprotocol (TurboCapture, Qiagen).

First-strand cDNA synthesis was carried using the ImProm II reversetranscription system (Promega) by adding reagents directly to the wellscontaining the immobilized mRNA. After reverse transcription, the wellsare washed 3 times with 100 ul 10 mM Tris-Cl buffer (pH 7.5). VH and VLgenes are amplify by PCR using a mix of slightly degeneratedIgG-specific primers (see below). The 80 ul PCR reaction consisted of0.2 uM of forward and reverse primer mixes, 1× precision buffer(Agilent), 0.2 mM dNTP and 5U TaqPlus precision (Agilent). Thethermocycler program was: 95° C. for 3 min and 34 cycles (95° C. for 40s, 62° C. for 30 s, and 72° C. for 50 s); 72° C. for 10 min; 4° C.storage.

PCR products were gel purified (Qiagen Gel extraction Kit) and yieldquantified using an ND-1000 spectrophotometer (Nanodrop). Illuminaadapters and compatible indices are then added by limited cycle PCR. The30 ul PCR reaction consisted of 20 ng PCR product (see above), 5 ul ofeach Nextera XT index primer (N7xx and N5xx) and 2× KAPA HiFi HotStartReady Mix. The thermocycler program was: 95° C. for 3 min and 8 cycles(98° C. for 20 s, 62° C. for 15 s, and 72° C. for 50 s); 72° C. for 10min; 4° C. storage. PCR products were gel purified (Qiagen Gelextraction Kit) and submitted for Illumina MiSeq 2×300 sequencing (UCDavis Genome Center).

VH Amplification Primers Forward: (SEQ ID NO: 1)5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATGGAGACTGGGCT GCGCTGGCTTCTCC-3′Reverse (1:1 ratio) (SEQ ID NO: 2)5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAGTGGGAAGACTG ACGGAGCCTTAG-3′(SEQ ID NO: 3) 5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAGTGGGAAGACTGATGGAGCCTTAG-3′ VK Amplification Primers Forward: (SEQ ID NO: 4)5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATGGACACGAGGGC CCCCAC-3′Reverse (9:0.5:0.5 ratio) (SEQ ID NO: 5)5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGTGGGAAGAKGA GGACAGTAGG-3′(SEQ ID NO: 6) 5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGTGGGAAGAKGAGGACACTAGG-3′ (SEQ ID NO: 7)5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGTGGGAAGAKGA GGACAGAAGG-3′

Example 4 Data Analysis Methods

Pre-Processing:

Demultiplexing of the pooled libraries (n=10-15 per sequencing run) andFastQ file generation was performed using MiSeq Reporter softwarepre-installed with the Illumina MiSeq instrument. FastQ files containssequencing and associated Phred quality score for each base pair. Weused NGS QC Toolkit, an open source software, to evaluate the overallperformance and quality of the NGS sequencing run (PLoS One. 2012;7(2):e30619). The paired-end reads contained in the Illumina R1(forward) and R2 (reverse) files are combined using PEAR(Bioinformatics. 2014 Mar. 1; 30(5):614-20) to reconstitute the VH or VKregion of the antibody repertoire. The following parameters were usedfor sequence assembly: Each sequence are trimmed at the occurrence oftwo consecutive base pair of a Phred quality score less than 20; theminimal overlap length equal 50 base pair or greater; the p-value of thealignment is greater than 0.0001; and the length of the assemblesequence greater than 300 base pair. The FASTQ file was converted toFASTA format using FASTX-toolkit, a collection of command line tools forShort-Reads FASTA/FASTQ files preprocessing(hannonlab.cshl.edu/fastx_toolkit/) and the sequences are translated inamino acid for further analysis. Sequences with a “STOP” codon withinthe ORF are filtered out the dataset. A curated dataset is created byparsing the sequences for identical amino acid sequences and filteringout sequence redundancy. The number of identical sequence is recorded ina log file to capture the frequency of reads for a particular VH/VLsequences, i.e. reads count.

Mapping of VH/VL Structural Domains:

The Kabat numbering scheme is a widely adopted standard for numberingthe amino acid residues within the variable light and heavy chain(Al-Lazikani et al., (1997) JMB 273, 927-948; summary:http://www.bioinf.org.uk/abs/). The Chothia numbering schema isidentical to Kabat, but places the insertion in CDR-L1 and H1 at thestructurally correct positions (Al-Lazikani et al., (1997) JMB 273,927-948). This is the numbering schema that we used for mappingstructural domains within the variable IgG region, i.e. delimitating theleader, FRM1, CDR1, FRM2, CDR2, FRM3, CDR3 and FRM4 domains. The VH/VLFramework 1-3 are generally highly conserved amino acid sequences, whilethe CDR1-3 are highly variable. A database was created based onapproximately 2300 know MAbs VH and VL sequences, which capture sequencevariations within the conserved Leader+FRM1, FRM2, FRM3 and FRM4. Thedatabases are then used as BLAST database to precisely delineate theLeader+FRM1, FRM2 and FRM3 of each sequences within the antibodyrepertoire NGS. Intervening amino acid sequences are assigned to thevariable CDR1, CDR2 and CDR3 domain, respectively.

Phylogenetic Pairing of Heavy and Light Chains:

A descending ordered list of VH or VL sequences is build based on thereads count determined at the pre-processing step. Frequency is aparticular IgG sequence is determined by a) the number of clonal B-cellspresent in the sample and, b) the average expression level of thisspecific sub-population. Therefore, a direct correlation can be seenbetween VH and VL sequence counts. The top most predominant sequencesare selected, in equal number in each ordered list, to construct anamino acid multiple alignment of the entire VH or VL region usingCLUSTALX2 (http://www.clustal.org/). The parameters for the multiplealignment are: open gap penalty=25; gap extension=0.2; delay divergentsequences=30%; and protein weight matrix=identity matrix. The outputunrooted trees was visualized using Dendroscope 3(http://ab.inf.uni-tuebingen.de/software/dendroscope/). Phylogenetictrees derived from VH and VK sequences are compared to each other'susing the tanglegram algorithm available in Dendroscope.

Example 5 Protocol for B-Cell Staining for Analysis by Flow Cytometry

Peripheral blood mononuclear cells (PBMCs) were isolated from a rabbitthat was immunized with keyhole limpet hemocyanin (KLH, PierceBiotechnology) and boosted two weeks prior to cell isolation with 0.5 mgKLH in phosphate buffered saline (PBS). Approximately five million ofthe purified PBMCs were incubated in media on a KLH coated petri dish(coated overnight with 1.5 ug/mL KLH in PBS, blocked with 10% fetalbovine serum (FBS), and washed two times with PBS) for 1.5 hours at 37°C. The cell coated petri dish was then washed three times with media andall non-adhering cells were collected and transferred to a newpolystyrene petri dish. The panned cells and the non-adhering cells werecultured in separate dishes for six days using the previously describedB-cell culture conditions. The resulting cell populations were thenharvested and stained for flow cytometry analysis.

For staining, approximately four million cells were collected from thepanned and the non-adhering cell cultures and resuspended for stainingin 400 uL 10% FBS in PBS. The cells were first surface stained for 20minutes at 4° C. with goat anti-rabbit IgG Fc specific Alexa Fluor 647conjugate (Jackson Immunoresearch, 1:100 dilution) and biotinylated KLH(prepared using Pierce Biotechnology EZ-Link Sulfo-NHS BiotinylationKit, 1 ug/100 uL). Following primary surface staining, the cells werewashed one time with 10% FBS in PBS and then incubated with streptavidinDylight 405 (Pierce Biotechnology, 1:200 dilution) for 10 minutes at 4°C. Next, the cells were washed two times with 10% FBS in PBS, fixedusing 2% paraformaldehyde in PBS at room temperature for 20 minutes, andthen washed one time with PBS. For intracellular staining, the sampleswere permeabilized using 0.25% saponin/10% FBS in PBS for 5 minutes,pelleted, and then stained with goat anti-rabbit IgG Fc specific AlexaFluor 488 conjugate (Jackson Immunoresearch, 1:100 dilution) andKLH-perCP (Abcam perCP conjugation kit, 1 ug/100 uL) at room temperaturefor 25 minutes in 400 uL of 0.25% saponin/10% FBS in PBS. Finally, thecells were washed two times with 0.25% saponin/10% FBS in PBS and storedin 1% paraformaldehyde in PBS until analysis.

Results Sequence Analysis

FIGS. 7A and 7B show a comparative analysis of VH sequences recoveredfrom the NGS of total PBMC versus B-cells after antigen specific panningand proliferation (BPP). Each column in FIGS. 7A and 7B represents aunique VH sequence with the abundance displayed on the Y-axis.Overlapping columns in FIG. 7A represent identical sequences recoveredfrom both datasets. FIG. 7B shows sequences recovered solely within theaffinity selected sample showing a significant increase in sequencingdepth for antigen-specific IgG.

Peripheral blood mononuclear cells were prepared from rabbit immunizedwith KDR protein as antigen. Two IgG libraries were prepared fromindependent bleeds. The first library was constructed from total B-cellspopulation (PBMC). The sample contains B-cells associated with theantigenic response as well as naïve B-cells in circulation 10 days afterthe last subcutaneous antigen injection (last antigenic boost). UniqueVH sequences are identified based of the full-length variable IgG domainand frequency for each reads above a threshold of 2 plotted in FIG. 7A(PBMC-RED). 50,513 unique VH sequences were identified by MiSeq 2×300sequencing.

The second VH library was constructed from affinity selected B-cells tothe KDR antigen (Affinity Selected—Blue). B-cells captured on solidsupport are then proliferated in-vitro in presence of CD40L, KDR antigenand cytokines, which promotes antigen-dependent memory B-celldifferentiation. In addition, only the B-cells initially activated byinteraction with the antigen differentiates into more mature b-cells.The transition of an antibody-presenting B-cells (memory B cells) intoan antibody-producing B-cells is accompany with a strong increase in IgGmRNA expression, which can be detected by NGS (read counts).

Unique VH sequences are identified from the dataset in association withtheir frequency (number of sequence reads) within the NGS dataset. Thedata is plotted in FIGS. 7A and 7B. The subset of Affinity-selectedsequences present in total PBMC is plotted alongside the correspondingPBMC sequences, illustrating that only a small fraction ofantigen-specific B-cells is present in total PBMC. 542 unique VHsequences are in common between PBMC and Affinity-selected/proliferatedB-cells (Affinity Selected—Blue). Approximately 1% of the unique VHsequences found in total PBMC correspond to the antigen-specificB-cells. As expected, mRNA abundance (number sequences reads) issignificantly larger in the affinity-selected VH than the correspondingfrequency in total PBMC.

As outlined above, affinity selection is providing a significantenrichment in antigen-specific B-cells. This results in an increase insequencing depth for KDR-specific B-cells and far greater representationwithin the dataset. FIG. 7B plotted unique VH sequences and theirabundance (sequence reads) recovered uniquely from the affinityselected/proliferated dataset. 41,923 additional unique VH sequenceswere recovered.

FIG. 9 shows that the sequences obtained from the enriched/activateddataset are highly polarized.

In next generation sequencing implementations of the method, no antibodyscreening is necessary. Using next generation sequencing one can samplethe full antigenic-response, which is represented by 1-5% of the totalB-cells in the PBMCs. This data shows that one can identifyantigen-specific sequence using NGS data without any screening and withhigh confidence. For example, the graph shown in FIG. 7A, plots VHsequences before (red) and after BPP (blue). The X-axis lists of all thenon-redundant VH sequences above a count of 2 within the datasets. TheY-axis represent the abundance of each sequence. The overlap isrepresented by sequences that present in both datasets. The nextgeneration sequencing approach can be used to capture the wholerepertoire of antigen-specific B cell with high resolution. This allowsone to apply rational approaches to the selection of candidateantibodies rather than simply relying on the “luck of the draw” providedby traditional methods. Moreover, abundance is a poor metric when usedas sole criteria to identify antigen-specific B-cells because, as shownin FIG. 7A, the most highly abundant sequences are, in fact, falsepositives.

By overlapping both sequence datasets (before enrichment andactivation=PBMC, after enrichment and activation), one can now identifywith high confidence which sequences are likely antigen-specific fromthe noise by looking at differential expression between the two datasets(blue line at least 2× above the red). This approach provides gooddiscrimination from noise.

Furthermore, when sequences recovered from B-cell cloning were comparedto the present data, one can see B-cell cloning results only results inthe most abundant antibodies. With the present method, we can capturethe entire spectrum of sequences, from high to low abundance.

Phylogenetic Pairing of Heavy and Light Chains

Lineage analysis was used to provide structure to the amino acidsequences derived for either VH or VL libraries sequence using theIllumina MiSeq 2×300. During affinity maturation, VH and VL chainsco-evolved through somatic hypermutation and gene conversion. Therelationship between related sequences can be capture by lineageanalysis of multiple alignments. A sorted list was created based onrelative abundance (read count) of each sequences within each VH and VLdataset. Read count relate on the clonal expansion (cell division)during the proliferation and expression level of mRNA encoding the VHand VL IgG. Any number a sequences can be used for the analysis. Forillustration purpose, we choose the first 100 most abundant unique VHand VL sequences from the ordered list. After multiple alignment usingCLUSTALX2, the phylogenetic tree is visualized in Dendroscope.

Defining co-linearity between the VH and VK lineage tree can bechallenging due to the diversity in sequences within the CDR1-3. Severaltools can be used to approximate VH/VL pairing. Abundance of individualVH and VL sequences was reported to help approximate pairing, but waslimited to the most abundant sequences within a highly polarized mousedataset. Local phylogenetic structures (relationship within related VHand VL sequences at multiple region of the tree) is another criteriathat can help aligning the whole trees but often only few are uniquelyshared across the phylogenetic trees.

Tanglegram Analysis

FIG. 8 shows a tanglegram of VH and VL sequences obtained using themethod described above. A total of 15 VH and VL sequences were linkedtogether using sequences that have a known pairing. As can be seen, thelinks between the trees are approximately parallel and there are nocross-overs. The approach shown in FIG. 8 relies on the mapping of knownVH/VL pairs derived from, e.g., antibodies that have been obtained in away that the VH and VL pairing is known to each VH and VL phylogenetictree as multiple anchors for co-alignment. This approach can be used inconjunction to abundance and local phylogenetic structure to increaseour confidence in the pairing of large dataset. With this method, atanglegram algorithm that programmatically resolved the bestco-alignment based on the “seed” sequences of known pairing was used.Additional pairs across the phylogenetic trees can then be selected forvalidation.

Characterization by Flow Cytometry

The cell population obtained from subjecting PBMCs of KLH-immunizedrabbits to the BPP procedure was characterized by flow cytometry. Fivemillion PBMC cells were used for B-cell panning and proliferation andthe flow through retained and cultured for comparison. Unlike in othermodel system, such as mouse or human, no surface marker antibodies areavailable to characterized B-cell population. However, crudeapproximation can be made by staining with for either the IgG-fc orantigen-specific B-cells.

Four million cells were collected from each culture and stained forintracellular IgG expression, characteristic for antibody producingB-cells, using anti-rabbit IgG-Fc-dylight 488 (FITC on graph).Similarly, the B-cell population producing KLH-specific antibody aredetected using KLH antigen conjugated with perCP (PI/PE-Cy5.5 on graph).FIG. 9 panel (A) shows that approximately 50% of the cells recoveredafter 7 days culture are expressing IgG (P2 sector, high FITC). Lessthan 10% of the cells in the flow-through stained for IgG, showing thatthe condition used for panning (37° C., no agitation followed by severalwashes) capture the large majority of the antigen-specific memoryB-cells. Gating of P2 (IgG+) and P3 (IgG−) population revealed that thelarge majority of the IgG+ B-cell are expressing antibodies interactingwith the KLH antigen (FIG. 9 panel (b)).

1. A method for producing an enriched population of antigen-specificplasma cells, comprising: (a) obtaining a sample of cells from an animalthat has been immunized by an antigen, wherein the sample comprises Bcells; (b) enriching for a population of antigen-specific B cells thatcomprise cell surface antibodies that are specific for the antigen by:i. contacting at least 10⁵ of the cells in said sample, en masse, withthe antigen or a portion thereof; and ii. isolating cells that bind tothe antigen or portion thereof; and (c) activating the enriched B cells,en masse, in the presence of the antigen or portion thereof, to producethe enriched population of antigen-specific plasma cells.
 2. The methodof claim 1, wherein the cells in the sample are obtained from thespleen, a lymph node, bone marrow or peripheral blood of the animal. 3.The method of claim 1, wherein the enrichment step (b) comprises: i.contacting at least 10⁵ of the cells in said sample, en masse, with asupport comprising the antigen, or a portion thereof, under conditionsby which said antigen-specific B cells bind to the antigen or portionthereof; and ii. washing the support to remove unbound cells.
 4. Themethod of claim 1, wherein the enrichment step (b) is done by i.contacting at least 10⁵ of the cells in said sample, en masse, with theantigen, or a portion thereof under conditions by which saidantigen-specific B cells bind to the detectably labeled antigen orportion thereof; and ii. sorting cells that bind to the antigen orportion thereof.
 5. The method of claim 1, further comprising: (d)fusing the enriched population of antigen-specific plasma cells with afusion partner to produce a plurality of hybridomas.
 6. The method ofclaim 5, further comprising (e) screening the hybridomas to identify ahybridoma that produces an antibody that binds to the antigen or portionthereof.
 7. The method of claim 1, further comprising (d) making cDNAfrom the enriched population of antigen-specific plasma cells; and (e)sequencing the cDNA to obtain a plurality of heavy chain variable domainsequences and a plurality of light chain variable domain sequences. 8.The method of claim 7, further comprising: (f) selecting a heavy chainsequence and a light chain sequence, and (g) testing an antibodycomprising the selected heavy and light chain sequences to determine ifthe antibody binds to the antigen or portion thereof.
 9. The method ofclaim 8, wherein the heavy and light chain sequences are selected by: i.obtaining a tanglegram of a plurality of the most abundant heavy andlight chain sequences, wherein the tanglegram is anchored using heavyand light chains that are naturally paired with one another; ii.selecting a heavy chain sequence and a light chain sequence, wherein theselected heavy and light chain sequences are aligned with one another inthe tanglegram; and iii. testing an antibody comprising the selectedheavy and light chain sequences to determine if the antibody binds tothe antigen or portion thereof.
 10. The method of claim 1, wherein theanimal is a rabbit, a mouse or a chicken.
 11. The method of claim 1,wherein the activating is done by CD40 activation.
 12. The method ofclaim 1, wherein the method further comprises collecting the enrichedpopulation of antigen-specific plasma cells, wherein the collecting isdone by collecting the medium from the support after the isolated Bcells are activated.
 13. The method of claim 1, wherein the enrichingstep enriches for antigen-specific B cells that compriseapplication-specific cell surface antibodies.
 14. The method of claim 1,wherein the method comprises: (a) obtaining a sample of cells from ananimal that has been immunized by an complex immunogen, wherein thecells comprise B cells; (b) enriching for a first population ofantigen-specific B cells that comprise cell surface antibodies that arespecific for a first antigen by: i. binding at least 10⁵ of the cells insaid sample, en masse, with the first antigen or a portion thereof; andii. isolating cells that bind to the first antigen or portion; and (c)activating the enriched B cells, en masse, in the presence of the firstantigen or portion thereof to obtain a first population ofantigen-specific plasma cells.
 15. The method of claim 14, wherein themethod comprises: (b) enriching for a second population ofantigen-specific B cells that comprise cell surface antibodies that arespecific for a second antigen of the immunogen by: i. binding at least10⁵ of the cells in said sample, en masse, with the second antigen or aportion thereof; and ii. isolating cells that bind to the second antigenor portion thereof; and (c) activating the enriched B cells, en masse,in the presence of the second antigen or portion thereof to obtain asecond population of antigen-specific plasma cells.